Rational Design of Fluorophores for In Vivo Applications - Progress in Molecular Biology and Translational Science - page 90

the solid-phase synthesis of nonsymmetrically substituted phthalocya-
nines. 220,221 This type of derivatives was previously prepared by statistical
condensation of different phthalonitriles, 222 which led to the formation of
complex mixture of products, diminished the yield of desired derivative,
and required extensive purification. Solid-phase synthesis

significantly

simplifies purification and improves the yield of nonsymmetrical

derivatives and enables an efficient synthesis of monofunctional,

bioconjugatable derivatives.

6. SPECIAL TYPES OF FLUOROPHORES
6.1. Fluorophores for multicolor imaging
Multicolor imaging (spectral multiplexing) allows targeting simultaneously
multiple different markers, processes, physicochemical parameters, cells, or
organs. Multicolor imaging requires access to a set of fluorophores in which
each fluorophore has a distinctive spectral feature so that fluorescence from
each of them can be independently detected in the presence of other
fluorophores. Ideally, each fluorophore in such a set should be excited with
the same wavelength, and each should exhibit a narrow emission band cen-
tered at a different wavelength, without overlap with emission bands from
the other fluorophores. Alternatively, each fluorophore should exhibit a
narrow absorption band (so that each can be selectively excited at the dif-
ferent wavelengths) and emit at the same wavelength.
Multicolor fluorescence detection has been successfully used, for exam-
ple, in nucleic acid sequencing (where energy-transfer dyads with a com-
mon donor and different acceptors have been used) 223,224 or in flow
cytometry. 225 Application of multicolor imaging in vivo has, however,
certain limitations. The simultaneous use of multiple organic fluorophores
in vivo is limited because of their broad emission bands (typical FWHM
for most organic fluorophores is > 30 nm) so that only a limited number
of fluorophores can be placed in the spectral window suitable for in vivo
applications without strong overlap of their emission bands. 6
Additionally, in the set of organic fluorophores with different emission
wavelengths, each fluorophore usually requires a different excitation
wavelength, which makes whole imaging process time consuming and
technically complex. Quantum dots, which have a tunable, narrow
emission band (FWHM 30 nm) and broad absorption bands that allow

excitations of the whole set of different quantum dots with the common
wavelength are good candidates for spectral multiplexing. 6 The use of the
quantum dots, however, raises concerns about their toxicity, as they often